Biomarkers of coronavirus pneumonia

ABSTRACT

The disclosure is directed to ABC294640, as free base or as salts thereof, in preparing medicines for treating coronavirus infection or preventing diseases caused by coronavirus infection, and a medicine for preventing coronavirus infection or preventing diseases caused by coronavirus infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/552,038, filed Dec. 15, 2021, which claims the benefit of andpriority to U.S. Provisional 63/125,427, filed Dec. 15, 2020, the entirecontent of each of which is hereby incorporated by reference in theirentirety.

BACKGROUND

Coronaviruses are lipid enveloped positive-stranded RNA viruses (+ ssRNA) that replicate in the cell cytoplasm. Prior to 2002, coronaviruseswere not considered to be significant human pathogens. Other humancoronaviruses such as HCoV-229E and HCoV-0C43 resulted in only mildrespiratory infections in healthy adults. In 2002, however, severe acuterespiratory syndrome coronavirus (SARS-CoV) emerged in GuangdongProvince, China. While SARS-CoV predominantly impacted Southeast Asia,with significant outbreaks throughout China, Hong Kong, Taiwan,Singapore, and Vietnam, the virus was carried outside the region.

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), wasdetected in a patient with severe respiratory disease in Saudi Arabia.The clinical features of MERS-CoV infection in humans range fromasymptomatic to very severe pneumonia with the potential development ofacute respiratory distress syndrome, septic shock, and multiorganfailure resulting in death. Since the first case of MERS-CoV infectionwas reported and the virus was isolated, significant progress has beenmade toward understanding the epidemiology, ecology, and biology of thevirus. Several assays for the detection of acute infection with MERS-CoVby real-time reverse transcription (RT)-PCR have been developed and arein widespread use.

In 2019, a novel coronavirus (nCoV) emerged in the world and is nowknown to cause coronavirus disease 2019 (COVID-19). COVID-19 is aninfectious disease caused by severe acute respiratory syndromecoronavirus 2 (SARS coronavirus-2 or SARS-CoV-2), a virusphylogenetically closely related to SARS virus. The World HealthOrganization (WHO) has declared the 2019-2020 coronavirus outbreak to bea Public Health Emergency of International Concern (PHEIC). For mostpatients, COVID-19 begins and ends in their lungs, because coronavirusesprimarily cause respiratory diseases.

SUMMARY

The present invention relates generally to the fields of virology,infectious disease and medicine. The invention provides a new use ofABC294640 as free bases or as salts thereof in the preparation ofmedicines for treating coronavirus infection in humans.

According to aspects illustrated herein, there is disclosed a method forthe treatment of a disease caused by a coronavirus, the methodcomprising confirming a patient is infected with a coronavirus and alsohas pneumonia; assessing that the patient requires supplemental oxygenat most at a fraction of inspired oxygen (FiO2) up to and including 60%;and administering to the patient an effective amount of ABC294640,

as a free base or as a salt thereof. In an embodiment, ABC294640 existsas a hydrochloride salt. In an embodiment, the ABC294640 is incombination with a pharmaceutically-acceptable carrier material. In anembodiment, the pharmaceutically-acceptable carrier material isphysiologically buffered saline. In an embodiment, a suspension isformed that includes ABC294640 hydrochloride suspended inphysiologically buffered saline and administering includes using a tubeto deliver the suspension directly to the stomach. In an embodiment, theABC294640 and optionally the pharmaceutically-acceptable carriermaterial, are in a unit dosage form suitable for oral administration. Inan embodiment, the unit dosage form is a solid dosage form. In anembodiment, the solid dosage form is a capsule. In an embodiment, thepatient is receiving oxygen via nasal cannula. In an embodiment, thecoronavirus is a respiratory syndrome coronavirus. In an embodiment, therespiratory syndrome coronavirus is a severe acute respiratory syndromecoronavirus. In an embodiment, the severe acute respiratory syndromecoronavirus is severe acute respiratory syndrome coronavirus-2(SARS-CoV-2). In an embodiment, the SARS-CoV-2 virus is wild-type. In anembodiment, the SARS-CoV-2 virus is a naturally occurring coronavirusvariant. In an embodiment, the unit dosage form suitable for oraladministration is a capsule having 250 mg of ABC294640 hydrochloride,and wherein administering includes two capsules administered twice aday, for at least 10 days, for a total daily dose of 1000 mg ofABC294640 hydrochloride.

According to aspects illustrated herein, there is disclosed a method forthe treatment of the 2019 coronavirus disease (COVID-19) caused by theSARS-CoV-2 virus, that includes administering to a person in needthereof an effective amount of ABC294640,

as a free base or as a salt thereof. In an embodiment, ABC294640 existsas a hydrochloride salt. In an embodiment, the ABC294640 is incombination with a pharmaceutically-acceptable carrier material. In anembodiment, the pharmaceutically-acceptable carrier material isphysiologically buffered saline. In an embodiment, a suspension isformed that includes ABC294640 hydrochloride suspended inphysiologically buffered saline and administering includes using a tubeto deliver the suspension directly to the stomach. In an embodiment, theABC294640 and optionally the pharmaceutically-acceptable carriermaterial, are in a unit dosage form suitable for oral administration. Inan embodiment, the unit dosage form is a solid dosage form. In anembodiment, the solid dosage form is a capsule. In an embodiment, theSARS-CoV-2 virus is wild-type. In an embodiment, the SARS-CoV-2 virus isa naturally occurring coronavirus variant. In an embodiment, the unitdosage form suitable for oral administration is a capsule having 250 mgof ABC294640 hydrochloride, and wherein administering includes twocapsules administered twice a day, for at least 10 days, for a totaldaily dose of 1000 mg of ABC294640 hydrochloride.

According to aspects illustrated herein, there is disclosed a method oftreatment comprising administering an effective amount of ABC294640,

as a free base or as a salt thereof, to a human having 2019 coronavirusdisease (COVID-19) caused by the SARS-CoV-2 virus. In an embodiment,ABC294640 exists as a hydrochloride salt. In an embodiment, theABC294640 is in combination with a pharmaceutically-acceptable carriermaterial. In an embodiment, the pharmaceutically-acceptable carriermaterial is physiologically buffered saline. In an embodiment, asuspension is formed that includes ABC294640 hydrochloride suspended inphysiologically buffered saline and administering includes using a tubeto deliver the suspension directly to the stomach. In an embodiment, theABC294640 and optionally the pharmaceutically-acceptable carriermaterial, are in a unit dosage form suitable for oral administration. Inan embodiment, the dosage form is a solid dosage form. In an embodiment,the solid dosage form is a capsule. In an embodiment, the SARS-CoV-2virus is wild-type. In an embodiment, the SARS-CoV-2 virus is anaturally occurring coronavirus variant. In an embodiment, the unitdosage form suitable for oral administration is a capsule having 250 mgof ABC294640 hydrochloride, and wherein administering includes twocapsules administered twice a day, for at least 10 days, for a totaldaily dose of 1000 mg of ABC294640 hydrochloride.

According to aspects illustrated herein, there is disclosed ABC294640,

as a free base or as a salt thereof, for use in treating coronavirusinfection.

According to aspects illustrated herein, there is disclosed ABC294640,

as a free base or as a salt thereof, for use in treating the 2019coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed(3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide), as a free base or as a salt thereof,for use in treating coronavirus infection.

According to aspects illustrated herein, there is disclosed(3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide), as a free base or as a salt thereof,for use in treating the 2019 coronavirus disease (COVID-19) caused bythe SARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed use ofABC294640,

as a free base or as a salt thereof, for the manufacture of a medicamentfor treatment of coronavirus infection.

According to aspects illustrated herein, there is disclosed use ofABC294640,

as a free base or as a salt thereof, for the manufacture of a medicamentfor treatment of the 2019 coronavirus disease (COVID-19) caused by theSARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed use of(3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide), as a free base or as a salt thereof,for the manufacture of a medicament for treatment of coronavirusinfection.

According to aspects illustrated herein, there is disclosed use ofcompound (3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide), as a free base or as a salt thereof,for the manufacture of a medicament for treating the 2019 coronavirusdisease (COVID-19) caused by the SARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of coronavirus infection,comprising ABC294640,

as a free base or as a salt thereof, and optionally apharmaceutically-acceptable carrier material.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of the 2019 coronavirusdisease (COVID-19) caused by the SARS-CoV-2 virus, comprising ABC294640,

as a free base or as a salt thereof, and optionally apharmaceutically-acceptable carrier material.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of coronavirus infection,comprising (3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide), as a free base or as a salt thereof,and optionally a pharmaceutically-acceptable carrier material.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of the 2019 coronavirusdisease (COVID-19) caused by the SARS-CoV-2 virus, comprising(3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide), as a free base or as a salt thereof,and optionally a pharmaceutically-acceptable carrier material.

According to aspects illustrated herein, there is disclosed ananti-coronavirus infection agent comprising ABC294640,

as a free base or as a salt thereof.

According to aspects illustrated herein, there is disclosed ananti-coronavirus infection agent comprising(3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide), as a free base or as a salt thereof.

According to aspects illustrated herein, there is disclosed a method forthe treatment of human coronavirus infection, comprising administeringto a subject in need thereof a therapeutically effective amount ofABC294640 (3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide) or a pharmaceutically acceptable saltthereof. In an embodiment, the method further comprises diagnosticallyconfirming that the subject is infected with a human coronavirus priorto administering ABC294640. In an embodiment, ABC294640 exists as ahydrochloride salt. In an embodiment, the coronavirus infection issevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

According to aspects illustrated herein, there is disclosed a method oftreating COVID-19 (SARS-CoV-2) coronavirus infection, the methodcomprising administering to a subject in need thereof for at least 10days one or more therapeutically effective doses of ABC294640(3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide) or a pharmaceutically acceptable saltthereof. In an embodiment, the method further comprises diagnosticallyconfirming that the subject is infected with SARS-CoV-2 prior toadministering ABC294640. In an embodiment, ABC294640 exists as ahydrochloride salt. In an embodiment, the total dose of ABC294640 perday is independently selected upon each occurrence from about 250 mg toabout 1500 mg.

According to aspects illustrated herein, there is disclosed a method fortreating COVID-19 (SARS-CoV-2) coronavirus infection, comprisingadministering to a human subject in need thereof a therapeuticallyacceptable amount of ABC294640(3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid(pyridin-4-ylmethyl)-amide) or a pharmaceutically acceptable saltthereof, ABC294640 having the ability to act on a host cell factor,sphingosine kinase-2 (SK2), which is involved in both viral replicationinside the cell and downstream inflammatory/immune responses.

According to aspects illustrated herein, there is disclosed a method ofmodulating replication of coronavirus in a host cell infected with thecoronavirus comprising administering to the host cell ABC294640 as afree base or as a salt thereof, in an amount effective to modulatereplication of the virus.

According to aspects illustrated herein, there is disclosed use ofABC294640 as a free base or as a salt thereof in the preparation ofdrugs for treating coronavirus infection. In an embodiment, thecoronavirus is a 2019 novel coronavirus COVID-19. In an embodiment, thecoronavirus infection is coronavirus pneumonia. In an embodiment,ABC294640 exists as a hydrochloride salt. In an embodiment, theABC294640 is active against a host cell factor, sphingosine inase-2,which is involved in both viral replication inside the cell anddownstream inflammatory/immune responses.

According to aspects illustrated herein, the present invention featuresa packaged pharmaceutical product. The packaged pharmaceutical productincludes a container, a plurality of ABC294640 unit dosage formssuitable for oral administration in the container, and a legend (e.g., alabel or an insert) associated with the container and indicatingadministration of ABC294640 for treating 2019 coronavirus disease(COVID-19) caused by the SARS-CoV-2 virus.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed embodiments will be further explained withreference to the attached drawings. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the presently disclosed embodiments.

FIG. 1 is a depiction of the human EpiAirway™ cell culture model, hereinreferred to as human bronchial epithelial cells (HBEC).

FIG. 2 . is a graph showing that in opaganib-treated,SARS-CoV-2-infected HBEC cultures, after 3 days incubation, show adose-dependent reduction in infectious virus production was observed atpharmacologically relevant concentrations.

FIG. 3 is a graph showing opaganib-treated, SARS-CoV-2-infected HBECcultures, after 3 days incubation, show limited cytotoxicity across thedose range where the potent anti-viral effects are seen.

FIG. 4 shows a Kaplan-Meier curve of time to no longer receivingsupplemental oxygen for at least 24 hours (mITT sensitivity) poststatistical analysis from the randomized, double-blind,placebo-controlled Phase 2a study of opaganib in COVID-19 pneumoniadescribed in Example 4.

FIG. 5 shows a Kaplan-Meier curve of time cumulative incidence for timeto 50% reduction from baseline in supplemental oxygen based on oxygenflow in L/min (mITT sensitivity) post statistical analysis from therandomized, double-blind, placebo-controlled Phase 2a study of opaganibin COVID-19 pneumonia described in Example 4.

FIG. 6 shows a dot plot of total supplemental oxygen requirement (areaunder the curve) for percent change from baseline using daily oxygenflow (L/min) measurements for 14 days (day 1 to day 14) post statisticalanalysis from the randomized, double-blind, placebo-controlled Phase 2astudy of opaganib in COVID-19 pneumonia described in Example 4.

FIG. 7 shows a Kaplan-Meier curve of time to death through end offollow-up (Day 42) for mITT with baseline FiO₂≤60% post statisticalanalysis from the randomized, double-blind, placebo-controlled Phase 2/3study of opaganib in COVID-19 pneumonia described in Example 5.

FIG. 8 shows a Kaplan-Meier curve of median time to discharge which was4 days shorter with opaganib vs. placebo (10 vs. 14) (251 patients fromthe study mITT population with baseline FiO₂<60%) post statisticalanalysis from the randomized, double-blind, placebo-controlled Phase 2/3study of opaganib in COVID-19 pneumonia described in Example 5.

DEFINITIONS

As used herein, the term “agent” refers to a drug substance havingpharmacological activity—an effect of the agent on an individual. Theterms “agent,” “active ingredient”, “drug substance,” and “compound” areused interchangeably herein.

As us herein, the term ABC294640 refers to[3-(4-chlorophenyl)-adamantane-1-carboxylic acid(pyridin-4-ylmethyl)amide] in a form as a free base or salt, or instereoisomeric or non-stereoisomeric form. In the case of compounds,salts, prodrugs or solvates that are solids, it is understood by thoseskilled in the art that the inventive compounds, salts, and solvates mayexist in different crystal forms, all of which are intended to be withinthe scope of the present invention. Opaganib, also known as ABC294640hydrochloride, is a specific salt form of ABC294640.

As used herein, the term “coronavirus” includes naturally occurring(e.g. wild-type) coronavirus; naturally occurring coronavirus variants;and coronavirus variants generated in the laboratory, including variantsgenerated by selection, variants generated by chemical modification, andgenetically modified variants (e.g., coronavirus modified in alaboratory by recombinant DNA methods). In an embodiment, a subject canbe tested for a viral infection within a few days after symptoms begin,or after treatment according to the present disclosure, by collectingnasal secretions (nasal or nasopharyngeal (NP) swabs), throat(oropharyngeal) swab, blood, or other body fluid samples and testing thesample for detection of viral antigens or RNA in blood and other bodyfluids using, for example, an antigen-capture enzyme-linkedimmunosorbent assay (ELISA), using an IgM ELISA (to determine whetherthe subject has IgM antibodies), using an IgG ELISA (to determinewhether the subject has IgG antibodies), using polymerase chain reaction(PCR), or by virus isolation. In an embodiment, the coronavirus isselected from the group consisting of Middle East respiratory syndrome(MERS), severe acute respiratory syndrome (SARS) and SARS-CoV-2.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

The terms “co-administer,” “coadministration,” or “in combination” areused to describe the administration of a compound of the presentinvention in combination with at least one other antiviral active agent.The timing of the coadministration is best determined by the medicalspecialist treating the patient. It is sometimes desired that the agentsbe administered at the same time. Alternatively, the drugs selected forcombination therapy may be administered at different times to thepatient. Of course, when more than one viral or other infection or othercondition is present, the present compounds may be combined with otheragents to treat that other infection or condition as required.

As related to the present invention, the term “treatment”, “treating”,and the like, is defined as prior to prophylactic administration of thecompounds in the methods described herein, prior to viral infection, orinhibiting viral activity after infection has occurred. In anembodiment, the term “treating” is meant to administer one or morecompounds of the present invention to measurably inhibit the replicationof a virus in vitro or in vivo, to measurably decrease the load of avirus in a cell in vitro or in vivo, or to reduce at least one symptomassociated with having a CoV-mediated disease in a patient. Desirably,the inhibition in replication or the decrease in viral load is at least10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, as determined using a suitable assay.Assays that monitor replication of viruses include, but are not limitedto, cytopathic viral assays, reporter-virus and reporter-cell assays,viral replicon assays, and gene-targeted viral assays. Viral loadtesting can be carried out using nucleic acid amplification based tests(NATs or NAATs) and non-nucleic acid-based tests on blood plasma samplesto determine the quantity of virus in a given volume including viral RNAlevels in plasma and tissue and total viral DNA. Alternatively, incertain embodiments, treatment is observed by a trained physician as anappreciable or substantial relief of symptoms in a patient with aCoV-mediated disease. Typically, a decrease in viral replication isaccomplished by reducing the rate of RNA polymerization, RNAtranslation, protein processing or modification, or by reducing theactivity of a molecule involved in any step of viral replication (e.g.,proteins or coded by the genome of the virus or host important for viralreplication). In an embodiment, the term “treat” refers to the abilityof a compound or compounds of the present invention to inhibit orsuppress replication of a virus, such as an RNA virus. In an embodiment,the term “treat” refers to the ability of a compound or compounds of thepresent invention to inhibit the cytopathic effect during a RNA virusinfection.

In some embodiments, an “effective amount” or “immune-stimulatoryamount” of a compound of the invention is an amount which, whenadministered to a subject, is sufficient to engender a detectable immuneresponse. In other embodiments, a “protective effective amount” of animmunogenic composition is an amount which, when administered to asubject, is sufficient to confer protective immunity upon the subject.In other embodiments, a “therapeutic effect amount” of a compound is anamount which, when administered to a subject, is sufficient to treat aviral infection, such as increase viral clearance.

As used herein, the term “fraction of inspired oxygen” or “FiO2” refersto an estimation of the oxygen content a person inhales, delivered insupplemental flow necessary to meet oxygenation goals, ideally targetinga peripheral oxygen saturation (SpO2) of between 90 and 96 percent.Typically <10 L/min will yield a FiO2 of up to 60%.

The agents and methods of the present invention may be utilized to treata subject in need thereof. In certain embodiments, the subject is amammal such as a human, or a non-human mammal. When administered to ananimal, such as a human, the agent is preferably administered as apharmaceutical composition comprising, for example, at least one agentof the invention with a substance or collection of substances capable ofbeing combined with the at least one agent. The term“pharmaceutically-acceptable carrier materials” as used herein means asubstance or collection of substances capable of being combined with anagent that is suitable for use in contact with the tissues of mammalsfor purposes of a therapeutic treatment in the mammals under anticipatedexposure conditions. Pharmaceutically-acceptable carrier materials arewell known in the art and include, for example, inert solid, semi-solidor liquid filler, diluent, encapsulating material.Pharmaceutically-acceptable carrier materials must, of course, be ofsufficiently high purity and sufficiently low toxicity to render themsuitable for administration to the human or lower animal being treated.The pharmaceutical composition can be in unit dosage form such astablet, capsule (including sprinkle capsule and gelatin capsule),granule, powder, syrup, suppository, injection or the like.

The term “immune response” refers to a response of a cell of the immunesystem, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, toa stimulus such as an antigen. An immune response can include any cellof the body involved in a host defense response, including for example,an epithelial cell that secretes an interferon or a cytokine. An immuneresponse includes, but is not limited to, an innate immune response orinflammation. As used herein, a protective immune response refers to animmune response that protects a subject from infection (preventsinfection or prevents the development of disease associated withinfection).

By “more effective” is meant that a treatment exhibits greater efficacy,or is less toxic, safer, more convenient, or less expensive than anothertreatment with which it is being compared. Efficacy may be measured by askilled practitioner using any standard method that is appropriate for agiven indication.

As used herein, the term “a suitable period of time” refers to theperiod of time starting when a patient begins treatment for a diagnosisof coronavirus infection using a method of the present disclosure,throughout the treatment, and up until when the patient stops treatmentdue to either a reduction in symptoms associated with the coronavirusinfection or due to a laboratory diagnosis indicating that the viralinfection is under control. In an embodiment, a suitable period of timeis one (1) week. In an embodiment, a suitable period of time is betweenone (1) week and two (2) weeks. In an embodiment, a suitable period oftime is two (2) weeks. In an embodiment, a suitable period of time isbetween two (2) weeks and three (3) weeks. In an embodiment, a suitableperiod of time is three (3) weeks. In an embodiment, a suitable periodof time is between three (3) weeks and four (4) weeks. In an embodiment,a suitable period of time is four (4) weeks. In an embodiment, asuitable period of time is between four (4) weeks and five (5) weeks. Inan embodiment, a suitable period of time is five (5) weeks. In anembodiment, a suitable period of time is between five (5) weeks and six(6) weeks. In an embodiment, a suitable period of time is six (6) weeks.In an embodiment, a suitable period of time is between six (6) weeks andseven (7) weeks. In an embodiment, a suitable period of time is seven(7) weeks. In an embodiment, a suitable period of time is between seven(7) weeks and eight (8) weeks. In an embodiment, a suitable period oftime is eight (8) weeks.

As used herein, the term “cytopathic effects” refers to the changes incell morphology due to a viral infection.

As used herein, the terms “cytopathogenesis” or “pathogenesis” includesinhibition of host cell gene expression and includes other cellularchanges that contribute to viral pathogenesis in addition to thosechanges that are visible at the microscopic level.

The term “in vitro” as used herein refers to procedures performed in anartificial environment, such as for example, without limitation, in atest tube or cell culture system. The skilled artisan will understandthat, for example, an isolate SK enzyme may be contacted with amodulator in an in vitro environment. Alternatively, an isolated cellmay be contacted with a modulator in an in vitro environment.

The term “in vivo” as used herein refers to procedures performed withina living organism such as, without limitation, a human, monkey, mouse,rat, rabbit, bovine, equine, porcine, canine, feline, or primate.

DETAILED DESCRIPTION

The disclosure relates generally to the fields of virology, infectiousdisease, and medicine and describes compounds, compositions, methods andkits for the treatment of Codi-mediated disease, e.g., one caused bySARS-CoV-2, BARS, or MERS. In an embodiment, the compositions compriseABC294640 and a pharmaceutically-acceptable carrier material. In anembodiment, the present disclosure provides a new use/application ofopaganib in the preparation of medicines for treating coronavirusinfection in humans.

More specifically, the invention relates to effective inhibitors ofcoronaviruses which can treat coronaviruses, including the 2019 novelcoronavirus. The invention provides a new use of opaganib as aneffective inhibitor of coronaviruses, and its application in thepreparation of drugs for treating coronavirus infection in humans.

ABC294640, [3-(4-chlorophenyl)-adamantane-1-carboxylic acid(pyridin-4-ylmethyl)amide], is an orally-administered, sphingosinekinase 2 (“SphK2” or “SK2”) inhibitor. ABC294640 is represented by thefollowing structural formula:

and can be prepared as a free base, in the form of its salts, andcrystalline modifications. U.S. Pat. Nos. 7,338,961, 8,063,248,8,324,237 and 8,557,800, which are incorporated herein by reference,teach these compounds, use, and methods of making same.

ABC294640 as the hydrochloride salt has been given an internationalnonproprietary name (INN) of opaganib and is represented by thefollowing structural formula:

The molecular formula of opaganib is C₂₃H₂₅ClN₂O.HCl with a molecularmass of 417.4 g/mol. Opaganib is a non-hygroscopic, white to off-whitesolid which is practically insoluble in water and ethyl acetate. In anembodiment, a medicine is prepared by filling opaganib in hard gelatinsize 1 capsules that further comprise at least onepharmaceutically-acceptable carrier material. In an embodiment, thepharmaceutically-acceptable carrier material are selected from thefollowing excipients: microcrystalline cellulose; colloidal silicondioxide; magnesium stearate vegetal; titanium dioxide. In an embodiment,opaganib capsules contain 250 mg ABC294640 as the hydrochloride salt or228.16 mg of ABC294640 free base. In an embodiment, opaganib capsulescontain 375 mg ABC294640 as the hydrochloride salt or 342.24 mg ofABC294640 free base.

In an embodiment, opaganib 250 mg capsules contain the agent ABC294640as the hydrochloride salt along with excipients that are encapsulated ingelatin, white opaque body and cap, coni-snap capsules, size 1. In anembodiment, opaganib 375 mg capsules contain the agent ABC294640 as thehydrochloride salt along with excipients that are encapsulated ingelatin, white opaque body and cap, coni-snap capsules, size 1.

Opaganib for treating coronavirus infection is generally administered inan amount ranging from about 250 mg to about 1500 mg per day. In anembodiment, opaganib 250 mg is administered as two capsules, twice perday, for a total daily dose of 1000 mg. In an embodiment, opaganib 250mg is administered as two capsules, 500 mg, Q12 hours. In an embodiment,a patient with a confirmed coronavirus infection is provided withinstructions to take a single 500 mg dose of opaganib (as two 250 mgcapsules) every 12 hours (so 1000 mg opaganib per day), for a total ofup to 2 consecutive weeks, or up to consecutive 14 days.

The inventors have discovered the new use of opaganib after a lot ofresearch. Opaganib has demonstrated antiviral, anti-inflammatory, andanti-thrombotic activity—acting on both the cause and the effects ofCOVID-19. Opaganib targets sphingosine kinase-2, a human cell componentinvolved in viral replication and not the virus itself. The mountingevidence of new SARS-CoV-2 mutations emerging globally underscores theimportance of this unique mechanism, which potentially minimizes therisk of viral resistance to therapy.

Provided herein are packaged pharmaceutical products, also known aspharmaceutical kits, that includes a container, a plurality of opaganibunit dosage forms suitable for oral administration in the container, anda legend (e.g., a label or an insert) associated with the container andindicating administration of opaganib for treating coronavirusinfection. In an embodiment, the legend includes instructions forcarrying out the methods described above and/or how to use the kit.Instructions included in the kit can be affixed as a label to packagingmaterial or can be included as a package insert. While instructions aretypically written or printed materials, they are not limited to such.Any medium capable of storing instructions and communicating them to anend user is contemplated by this disclosure. Such media include, but arenot limited to, electronic storage media (e.g., magnetic discs, tapes,cartridges), optical media (e.g., CD ROM), and the like. As used herein,the term “instructions” can include the address of an internet sitewhich provides instructions.

Combination and Alternation Therapy

The compounds described herein can be administered on top of the currentstandard of care for COVID patients, or in combination or alternationwith any other compound or therapy that the healthcare provider deemsbeneficial for the patient. The combination and/or alternation therapycan be therapeutic, adjunctive, or palliative. When the methods includeadministering to a patient more than one active agent, the agents may beadministered within 7, 6, 5, 4, 3, 2 or 1 days; within 24, 12, 6, 5, 4,3, 2 or 1 hours, within 60, 50, 40, 30, 20, 10, 5 or 1 minutes; orsubstantially simultaneously. The methods of the invention may includeadministering one or more agents to the patient by oral, systemic,parenteral, topical, intravenous, inhalational, or intramuscularadministration.

It has been observed that COVID patients can pass through various stagesof disease, and that the standard of care can differ based on what stageof illness the patient presents with or advances to. COVID is noteworthyfor the development of “cross-talk” between the immune system and thecoagulation system. As the disease progresses, the patient can mount anoverreaction by the immune system, which can lead to a number of seriousimplications, including a cytokine storm. Via the cross-talk between theimmune system and the coagulation system, the patient can begin clottingin various areas of the body, including the respiratory system, brain,heart and other organs. Multiple clots throughout the body have beenobserved in COVID patients, requiring anticoagulant therapy. It isconsidered that these clots may cause long term, or even permanentdamage if not treated and disease alleviated.

More specifically, COVID-19 has been described as progressing throughthree general stages of illness: stage 1 (early infection), stage 2(pulmonary phase), and stage 3 (hyperinflammation phase/cytokine storm).

Stage 1 is characterized by non-specific, and often mild, symptoms.Viral replication is occurring, and it is appropriate to begin immediatetreatment with the compounds described herein and perhaps in combinationor alternation with another anti-viral therapy. Interferon-β may also beadministered to augment the innate immune response to the virus. In oneembodiment, therefore, a compound of the present invention is used in aneffective amount in combination or alternation with interferon-β and oran additional anti-viral drug. Zinc supplements and or Vitamin C is alsosometimes administered at this stage or as the illness progresses.

Stage 2 of COVID-19 is the pulmonary phase where patients may experienceacute hypoxemic respiratory failure. In fact, the primary organ failureof COVID-19 is hypoxemic respiratory failure. It has been shown thatmoderate immunosuppression via a steroid, for example, dexamethasone,can be beneficial to patients with acute hypoxemic respiratory failureand/or patients on mechanical ventilation. In one embodiment, a compoundthe present invention is used in an effective amount in combination witha corticosteroid which may be a glucocorticoid. Non-limiting examplesare budesonide (Entocort EC), bethamethasone, (Celestone), prednisone(Prednisone Intensol), prednisolone (Orapred, Prelone), triamcinolone(Aristospan Intra-Articular, Aristospan Intralesional, Kenalog),methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), hydrocortisone,or dexamethasone (Dexamethasone Intensol, DexPak 10 Day, DexPak 13 Day,DexPak 6 Day).

The NS5B inhibitor Remdesivir has provided mixed results when given toCOVID19 patients. It can only be administered in a hospital setting, andonly by intravenous injection, typically three times a day, which makesit inappropriate for mild to moderate COVID19 patients. In oneembodiment, a compound of the present invention is administered incombination or in alternation with Remdesivir to amplify the overallantiviral effect.

Stage 3, the final stage of the disease, is characterized by progressivedisseminated intravascular coagulation (DIC), a condition in which smallblood clots develop throughout the bloodstream. This stage also caninclude multi-organ failure (e.g. vasodilatory shock, myocarditis). Ithas also been observed that many patients respond to this severe stageof COVID-19 infection with a “cytokine storm.” There does appear to be abi-directional, synergistic relationship between DIC and cytokine storm.To combat DIC, patients are often administered an anti-coagulant agent,which may, for example, be an indirect thrombin inhibitor or a directoral anticoagulant (“DOAC”). Non-limiting examples are low-molecularweight heparin, warfarin, bivalirudin (Angiomax), rivaroxaban (Xarelto),dabigatran (Pradaxa), apixaban (Eliquis), or edoxaban (Lixiana). In oneembodiment, a compound of the present invention is administered incombination or in alternation with anti-coagulant therapy. In somesevere cases of clotting in COVID patients, TPA can be administered(tissue plasminogen activator).

It has been observed that high levels of the cytokine interleukin-6(IL-6) are a precursor to respiratory failure and death in COVID-19patients. To treat this surge of an immune response, which mayconstitute a cytokine storm, patients can be administered anIL-6-targeting monoclonal antibody, pharmaceutical inhibitor or proteindegrader such as a bispecific compound that binds to IL-6 and also to aprotein that mediates degradation. Examples of antibodies includetocilizumab, sarilumab, siltuximab, olokizumab and clazakizumab. In oneembodiment, a compound of the present invention is administered incombination or in alternation with tocilizumab or sarilumab. Additionalnonlimiting examples of immunosuppressant drugs used to treat theoverreacting immune system include Janus kinase inhibitors (tofacitinib(Xeljanz)); calcineurin inhibitors (cyclosporine (Neoral, Sandimmune,SangCya)), tacrolimus (Astagraf XL, Envarsus XR, Prograf)); mTORinhibitors (sirolimus (Rapamune), everolimus (Afinitor, Zortress)); and,IMDH inhibitors (azathioprine (Azasan, Imuran), leflunomide (Arava),mycophenolate (CellCept, Myfortic)). Additional antibodies and biologicsinclude abatacept (Orencia), adalimumab (Humira), anakinra (Kineret),certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi),infliximab (Remicade), ixekizumab (Taltz), natalizumab (Tysabri),rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra),ustekinumab (Stelara), vedolizumab (Entyvio), basiliximab (Simulect),and daclizumab (Zinbryta)).

IL-1 blocks the production of IL-6 and other proinflammatory cytokines.COVID patients are also sometimes treated with anti-IL-1 therapy toreduce a hyperinflammatory response, for example, an intravenousadministration of anakinra. Anti-IL-1 therapy generally may be forexample, a targeting monoclonal antibody, pharmaceutical inhibitor orprotein degrader such as a bispecific compound that binds to IL-1 andalso to a protein that mediates degradation.

Patients with COVID often develop viral pneumonia, which can lead tobacterial pneumonia. Patients with severe COVID-19 can also be affectedby sepsis or “septic shock”. Treatment for bacterial pneumonia secondaryto COVID or for sepsis includes the administration of antibiotics, forexample a macrolide antibiotic, including azithromycin, clarithromycin,erythromycin, or roxithromycin. Additional antibiotics includeamoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin,metronidazole, sulfamethoxazole, trimethoprim, amoxicillin, clavulanate,or levofloxacin. In one embodiment, thus a compound of the presentinvention, is administered in combination or in alternation with anantibiotic, for example, azithromycin. Some of these antibiotics such asazithromycin have independent anti-inflammatory properties. Such drugsmay be used both as anti-inflammatory agents for COVID patients and havea treatment effect on secondary bacterial infections.

A unique challenge in treating patients infected with COVID-19 is therelatively long-term need for sedation if patients require mechanicalventilation which might last up to or greater than 5, 10 or even 14days. For ongoing pain during this treatment, analgesics can be addedsequentially, and for ongoing anxiety, sedatives can be addedsequentially. Non-limiting examples of analgesics include acetaminophen,ketamine, and PRN opioids (hydromorphone, fentanyl, and morphine).Non-limiting examples of sedatives include melatonin, atypicalantipsychotics with sedative-predominant properties (olanzapine,quetiapine), propofol or dexmedetomidine, haloperidol, andphenobarbital. In one embodiment, a compound of the present invention isadministered in combination or in alternation with a pain reliever, suchas acetaminophen, ketamine, hydromorphone, fentanyl, or morphine. In oneembodiment, a compound of the present invention is administered incombination or in alternation with a sedative, such as melatonin,olanzapine, quetiapine, propofol, dexmedetomidine, haloperidol, orphenobarbital.

Investigational drugs for COVID-19 include chloroquine andhydroxychloroquine. In one embodiment, a compound of the presentinvention, is administered in combination or in alternation withchloroquine or hydroxychloroquine.

A protease inhibitor such as lopinavir or ritonavir, previously approvedfor HIV, may also be administered.

Additional drugs that may be used in the treatment of a COVID patientinclude, but are not limited to favipiravir, fingolimod (Gilenya),methylprednisolone, bevacizumab (Avastin), Actemra (tocilizumab),umifenovir, losartan and the monoclonal antibody combination of REGN3048and REGN3051 or ribavirin. Any of these drugs or vaccines can be used incombination or alternation with an active compound provided herein totreat a viral infection susceptible to such.

In one embodiment, a compound of the present invention is used in aneffective amount in combination with anti-coronavirus vaccine therapy,including but not limited to mRNA-1273 (Moderna, Inc.), AZD-1222(AstraZeneca and University of Oxford), BNT162 (Pfizer and BioNTech),CoronaVac (Sinovac), NVX-CoV 2372 (NovoVax), SCB-2019 (Sanofi and GSK),ZyCoV-D (Zydus Cadila), and CoVaxin (Bharat Biotech). In anotherembodiment, a compound of the present invention is used in an effectiveamount in combination with passive antibody therapy or convalescentplasma therapy.

In an embodiment, a compound of the present invention is used in aneffective amount in combination with a 5-HT receptor antagonists, whichcan relieve certain symptoms that might be present in a patient infectedwith coronavirus, such as diarrhea.

SARS-CoV-2 is constantly mutating, which many increase virulence andtransmission rates. Drug-resistant variants of viruses may emerge afterprolonged treatment with an antiviral agent. Drug resistance may occurby mutation of a gene that encodes for an enzyme used in viralreplication. The efficacy of a drug against an RNA virus infection incertain cases can be prolonged, augmented, or restored by administeringthe compound in combination or alternation with another, and perhapseven two or three other, antiviral compounds that induce a differentmutation or act through a different pathway, from that of the principledrug.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples. The following examples are given for the purposeof illustrating various embodiments of the invention and are not meantto limit the present invention in any fashion.

EXAMPLES Example 1: Assessment of the Anti-Viral Activity of ABC294640Against SARS-CoV-2 in Human Airway Epithelial Cells

We designed an in vitro assessment in an organotypicair-liquid-interface (ALI) culture of human primary bronchial epithelialcells (HBEC; EpiAirway™, MatTek) to evaluate whether infection andspread of SARS-CoV-2 could be directly inhibited by opaganib. This humancell culture model system was selected because it contains apseudostratified epithelial layer that morphologically and functionallyresembles that of the human airway, consisting of ciliated and goblet(mucus producing) cells exposed to the air from the apical layer. Thesecells act as the first line of defense against invading viruses andserve as replication sites. Available evidence also suggests that humanbronchial epithelial cells express the host factors targeted by opaganib(sphingosine kinase-2).

Test Compounds: Opaganib—Test Compound

-   -   Description: Opaganib        [3-(4-chlorophenyl)-adamantane-1-carboxylic acid        (pyridin-4-ylmethyl) amide, hydrochloride salt] is an orally        available inhibitor of the enzyme sphingosine kinase-2 (SK2).        Solvent: DMSO

Remdesivir (GS-5734)—Positive Anti-Viral Control

-   -   Description: Remdesivir is a nucleotide-analog anti-viral        prodrug. It exhibits anti-viral activity against multiple        variants of EBOV with EC50 value ranging in 0.06-0.14 μM in        cell-based assays and broad-spectrum anti-viral activity in        vitro against other pathogenic RNA viruses, including SARS-CoV.        Solvent: DMSO 100 mg/mL (166.0 mM)        Bleomycin (sulfate)—Positive Cytotoxic Control    -   Description: Bleomycin is a chemotherapy agent commonly used for        the treatment of Hodgkin's lymphoma and embryonal carcinomas. A        broad spectrum of bleomycin-induced pulmonary toxicities have        been well described as a complication of such therapy, the most        common variant of which is bleomycin-induced pneumonitis (BIP)        (Sleijfer et al., 2001). Bleomycin (BLM) is chosen as the        best-studied micronucleus (MN) inducers in human lymphocytes        with different mechanisms of genotoxicity. Solvent: DMSO 16.67        mg/mL (11.2 mM)

Methods: Cell Culture—Differentiated Human Bronchial Epithelial Cells(HBEC)

Normal human bronchial epithelial (HBEC) cells were differentiated byMatTek Corporation (Ashland, Mass.) and arrived in kits with either 12-or 24-well inserts each. HBEC cells were grown on 6 mm{circumflex over( )}2 mesh disks in transwell inserts. Three days prior to shipment, thetissues were transferred into hydrocortisone-free medium. Duringtransportation the tissues were stabilized on a sheet of agarose, whichwas removed upon receipt. One insert was estimated to consist ofapproximately 1.2×10⁶ cells. Kits of cell inserts (EpiAirway™ AIR-100)originated from a single donor, #9831, a 23-year old, healthy,non-smoking, Caucasian male. The cells have unique properties in forminglayers, the apical side of which is exposed only to air and that createsa mucin layer. Upon arrival, the cell transwell inserts were immediatelytransferred to individual wells of a 6-well plate according tomanufacturer's instructions, and 1 mL of MatTek's proprietary culturemedium (AIR-100-MM) was added to the basolateral side, whereas theapical side was exposed to a humidified 5% CO₂ environment. Cells werecultured at 37° C. for one day before the start of the experiment. Afterthe 16-18 h equilibration period, the mucin layer, secreted from theapical side of the cells, was removed by washing with 400 μL pre-warmedTEER buffer. Culture medium was replenished following the wash step. Adepiction of the culture inserts and EpiAirway tissue provided in FIG. 1.

Treatment with Test Compounds:

Test compounds were serially diluted from stock solution (containingDMSO) in Assay medium (AIR-ASY-100, MatTek) and placed at roomtemperature. Test compound dilutions are outlined below (finalDMSO<0.5%). HBEC cultures were washed with phosphate-buffered saline(PBS) and incubated at 37° C. with remdesivir (2 μM), bleomycin sulfate(75.6 and 151 μg/ml) and opaganib (6 concentrations ranging from 0.05 to11.25 μg/ml) diluted in assay medium (AIR-100-ASY, MatTek) for 1 h priorto infection. For control wells, assay medium with DMSO (finalDMSO<0.5%; control) and virus only control (assay medium only) wereadded for the 1 h before infection. Compounds were added to each inserton the apical layer (0.15 mL) and basal layer (0.85 mL) in triplicate.

Viral Infection and Sample Processing:

After 1 hr incubation with compounds, the apical side of the cultureswere washed and then infected with SARS-CoV-2 clinical isolate(2019-nCoV/USA-WA1/2020) at MOI=0.1 PFU/cell for 1 h at 37° C., in thepresence of compound or assay control media. After 1 hr viralincubation, the virus was removed from the apical side, and cultureswere washed one time with PBS to remove any unbound virus. The cultureswere then incubated at 37° C. for 72 h with fresh compound. At 24 h and48 h post-infection, the basolateral medium was replaced with 1 mL offresh medium containing the respective compounds.

At 72 hours post-infection, tissues and media were collected forprocessing. The apical layer was washed with 0.4 mL of TEER buffer (PBSwith Mg²⁺ and Ca²⁺) and collected for viral titer assessment via TCID50(50% tissue culture infectious dose) assay. Eight-fold serial dilutionsof apical layer supernatant sample concentrations were added to 96-wellassay plates containing Vero E6 cells (20,000/well). The plates wereincubated at 37° C., 5% CO₂ and 95% relative humidity. Following 3 days(72±4 h) incubation, the plates were stained with crystal violet tomeasure cytopathic effect (CPE). Virus titers were calculated using themethod of Reed and Muench (Reed et al., 1938). The TCID₅₀ values weredetermined from triplicate samples.

To evaluate the health of HBEC cells after exposure to opaganib, controlcompounds, and viral infection, a lactate dehydrogenase (LDH) releaseassay was conducted. Medium from the basolateral layer of the tissueculture inserts was removed 72 hours post-infection and diluted in LDHStorage Buffer as per the manufacturer's instructions (Promega). Sampleswere further diluted with LDH Buffer and incubated with an equal volumeof LDH Detection Reagent. Luminescence was recorded after 60 minutesincubation at room temperature. A no cell control was included as anegative control to determine culture medium background and bleomycinincluded as a positive cytotoxic control. Luminescence was reported,with background levels found within the acceptable luminescence range(range 1,000-10,000).

Additionally, the apical layer of the HBEC tissues were collected byadding Trizol LS (Invitrogen) to each culture insert and pipetting upand down several times to lyse and collect the cells and store at −80°C. for future RNA and protein expression analysis.

Results: Opaganib, is Highly Active Against SARS-CoV-2 in HBEC Cultures

In this study, normal human bronchial epithelial cells (HBEC) werepretreated in triplicate with 6 different concentrations of opaganib(ranging from 11.25 to 0.05 μg/ml) both on the apical and basolateralside of each culture. Once pretreated, HBEC were exposed to SARS-CoV-2(2019-nCoV/USA-WA1/2020) for 1 h, the apical layer was washed to removeunbound virus, and the culture then incubated for 3 days with compound.At 3 days post infection, the apical layer was washed and assessed forviral load by TCID50 assay. The basolateral media was collected andassessed for presence of lactate dehydrogenase (LDH), which is releasedfrom damaged cells serving as an indicator of cell death/viability.

Opaganib demonstrated potent anti-viral activity, with viral replicationbeing inhibited in a dose-dependent manner without significantcompromise to cell viability. In opaganib-treated, SARS-CoV-2-infectedHBEC cultures, after 3 days incubation, a dose-dependent reduction ininfectious virus production was observed with complete inhibitionstarting at opaganib 1 μg/ml (a pharmacologically relevantconcentration). These results compare favorably with remdesivir, thepositive control in the study. Cell viability, as assessed in the LDHrelease assay, To demonstrate the anti-viral activity of opaganibagainst SARS-CoV-2 in a human primary epithelial culture system, weperformed anti-viral assays in HBEC cultures, which are grown onair-liquid interface and recapitulate the cellular complexity andphysiology of the human conducting airway. In opaganib-treated,SARS-CoV-2-infected HBEC cultures, after 3 days incubation, adose-dependent reduction in infectious virus production was observedwith complete inhibition starting at opaganib 1 μg/ml (apharmacologically relevant concentration) (FIG. 2 ). These resultscompare favorably with remdesivir, the positive control in the study.

Opaganib did not cause cytotoxicity in HBEC cultures across theconcentration range where the potent anti-viral effects are seen (FIG. 3). Together, these data demonstrate that opaganib is potently anti-viralagainst SARS-CoV-2 in primary human lung cultures without compromisingcell membrane integrity, a measure of cell viability and drug safety,further demonstrating opaganib's promising potential for treatingpatients with COVID-19.

At the concentration range tested, neither 50% inhibition nor 50%cytotoxicity were reached. At the lowest concentration tested, 0.05μg/ml, greater than 90% inhibition of infectious virus production wasreached. At highest concentration tested, 11.25 μg/ml, the cellsremained viable throughout the experiment. When utilizing the high andlow concentration range from this experiment to calculate theselectivity index (SI), the ratio that measures the window betweencytotoxicity and antiviral activity by dividing the antiviral activityvalue (AVA) into the toxicity (TOX) value (AVA/TOX), the SI value is225. The SI value is expected to be greater if a wider range ofconcentrations are tested.

Example 2: Assessment of In Vivo Efficacy of ABC294640 Against ARDSInduced Thrombosis

This study assessed the efficacy of ABC294640 to reduce the incidence ofadverse thromboembolic events in situ in acute respiratory distresssyndrome (ARDS) conditions using a rat venous stasis model. This assayis designed to measure thrombotic risks following LPS-induced lunginjury. LPS-induced lung injury is one of the most commonly used rodentmodels for ARDS and was described to mimic the neutrophilic inflammatoryresponse observed in ARDS patients. The mechanism of LPS-induced ARDS isbased on damage to endothelial cells and a systemic inflammatoryresponse.

The venous stasis (Wessler) test in animals has been used extensivelyfor over 40 years as a laboratory measure for in vivohypercoagulability. It has proved invaluable for assessing thethrombogenicity of various blood products.

Test compound was administered by oral gavage 3 hours post-instillationand 24, 48 and 72 hours post-instillation, at a dose of 250 mg/kg. Theappropriate amount of LPS from E. coli (O55:B5) was diluted in saline toobtain a final concentration of 400 μg/mL. This solution was given byintratracheal instillation (0.5 mL/kg). The vehicle was composed of PBSpH 7.4±0.1. ABC294640 was weighed and transferred in vehicle (PBS,0.375%, pH 7.4) to obtain a final solution at 25 mg/mL. ABC294640solution is stirred for 10 minutes at room temperature prior to dosing.This solution was given by oral gavage (250 mg/kg, 10 mL/kg).

Sprague-Dawley rats (male) weighing between 275 and 400 g were used forthis study. Animals were randomly assigned to a treatment group by theStudy Director. Food and water were given ad libitum. Observation forbehavior and general health status were done until the sacrifice. Thebody weight was recorded before the instillation and 24, 48 and 72 hourspost-instillation.

Arterial oxygen saturation (SpO₂) and heart rate was recorded with amouse pulse oximeter collar probe installed on the conscious mouse(MouseOx Plus system, Starr Life Sciences) before instillation and 24,48 and 72 hours post-instillation. Rats were also introduced into theplethysmograph chamber environment, same schedule as SpO₂. Thefunctional respiratory parameters were assessed by the whole-bodyplethysmograph (VivoFlow, SCIREQ). The functional respiratory parametersanalyzed included; the respiratory rate, the PenH (pulmonary congestionindex) and the inspiratory/expiratory time measurements. A blood samplewas also taken prior terminal procedure for complete blood count andcytokines level evaluation.

In this study, ARDS was induced by intratracheal instillation of LPS.Throughout the ARDS induction and development process, animal was dosedby oral gavage with vehicle or ABC294640 (3 h, 24 h, 48 and 72 hourspost-instillation). Following 72 hours conscious measurements, rats wereanesthetized and a venous stasis was performed on the inferior vena cava(4 hours post-gavage of 72 hour time point). The stasis was maintainedfor 30 minutes. The segment was then excised and its content scored.Subsequently, animal was euthanized by exsanguination.

After exsanguination, a tracheotomy was performed and the thoraciccavity opened to expose the lungs. The trachea was then connected to thecannula of a perfusion system. The left lung clamped while cold PBS 1×,Protease Inhibitor 1× solution was injected, by the trachea to perform abronchoalveolar lavage fluid (BALF) on the right lobes of the lungs andwas collected for further analysis. The total cells count with cellsdifferential count and the total protein content was assessed in theBALF samples. An aliquot of the BALF was kept for the quantification ofthe chemokines/cytokine's levels in BALF.

The left lobe of the lungs was excised. The left lobe freshly harvestedwas weighed wet to determine the left lung weight and left lung index(left lung weight/body weight×100). The lower part of the left lobe wasused to determine the left lung wet/dry ratio, an indicator of pulmonaryedema. The remaining part of the left lung was homogenized and aliquotedfor the quantification of protein content.

Induction of LPS Lung Injury:

-   -   1. Prior LPS or saline instillation, the functional respiratory        parameters of all rat was assessed by whole-body plethysmography        and SpO₂ was evaluated on conscious rat with a collar probe        pulse oximeter. Rat was first acclimatized to the plethysmograph        chamber prior to the physiological assessment. The respiratory        rate, the Penh and the inspiratory/expiratory time measurements        was analyzed.    -   2. Rats were anaesthetized with 2.5% isoflurane USP (Abbot        Laboratories, Montreal Canada) in oxygen. Rats were then        intubated and LPS or saline delivered by intratracheal        instillation.    -   3. Rats recovered from anesthesia and returned to their        respective cage.    -   4. Three hours post instillation, the first dose of ABC294640        was administered by oral gavage (see Table 1 below).

TABLE 1 Experimental Progression/Steps for Rats Gavage Dose Volume GroupInstillation Test compound Composition (mg/kg) (mL/kg) 1 Saline Vehicle0.9% NaCl N/A 10 2 LPS Vehicle 0.9% NaCl N/A 10 3 LPS ABC294640 25 mg/mL250 10

-   -   5. Rats were evaluated periodically to ensure animal well-being        (general behavior and daily body weight).    -   6. Animals were also dosed by oral gavage 24, 48 and 72 hours        post-instillation.    -   7. SpO₂ and whole-body plethysmography were also evaluated 24,        48 and 72 hours post-instillation.    -   8. A blood sample was withdrawn from jugular vein prior venous        stasis procedure for complete blood count and cytokines level        measurements.    -   9. Four hours following the last dosing, rats were anaesthetized        with 2.5% isoflurane USP (Abbot Laboratories, Montreal Canada)        in oxygen. The procedure was performed on a homeothermic blanket        to control body temperature.    -   10. The rat's inferior vena cava was exposed and two (2) loose        sutures were placed 1 cm apart. Any collateral vessels of the        isolated segment were ligated.    -   11. Stasis was maintained in situ for a period 30 minutes.    -   12. The venous stasis segment was removed, opened        longitudinally, emptied on a filter paper and photographed. Any        existing thrombi was removed and blotted on a filter paper. The        thrombi was measured, weighed and scored on a scale from 0 to 4        (see Table 2).

TABLE 2 Quantitative Evaluation of Thrombogenicity Quantitativeevaluation of thrombogenicity Score No clot 0 Few macroscopic strands offibrin are barely visible 0.5 Few macroscopic strands of fibrin 1.0 Oneor several thrombi ≤1.5 mm 1.5 One thrombi ≥1.5 mm 2.0 Two or morethrombi ≥1.5 mm 2.5 One large thrombus ≥3 mm 3.0 Two or more largethrombi ≥3 mm 3.5 Single Thrombus forming the whole segment 4.0

-   -   13. Following venous stasis, the animals were euthanized by        exsanguination and bronchoalveolar lavage was collected from the        right lung. To do so, the muscle over the trachea was dissected        away prior to performing a tracheotomy. The thoracic cavity was        opened to expose the lungs and the trachea was connected to the        cannula of a perfusion system. The left lung was clamped while        15 mL (3×5 mL) of cold PBS 1×, Protease Inhibitor 1× solution        was injected by the trachea to perform a bronchoalveolar lavage        fluid (BALF) on the right lobe of the lungs. BALF was collected        for further analysis. The total cells count with cells        differential count was assessed in the BALF samples. An aliquot        of the BALF was kept for quantification of the        chemokines/cytokines levels in BALF.    -   14. The left lung was then harvested and weighed for left lung        weight and left lung index calculation. Lung tissue edema was        assessed using wet/dry ratio calculation. The lower part of the        left lung was weighted alone (wet weight) and used to determine        the lung wet/dry ratio. Following drying at 60° C. for at least        24 hours, it was reweighed (dry weight).

Each parameter (listed below) were compiled for each group and presentedin bar graphs with appropriate statistical analysis.

-   -   1—Change in body weight    -   2—Saturation (SpO₂) and Heart Rate (bpm)    -   3—Respiratory parameters: Inspiratory/Expiratory Time        -   Tidal and expired volume        -   Respiratory Rate        -   PenH    -   4—BALF total cell count with cells differential    -   5—BALF Cytokines/Chemokines Levels    -   6—Left Lung Weight and index    -   7—Lung wet/dry ratio    -   8—Lung total protein content in the lung homogenate

LPS induces a significant increase in lung weight associated with theinflammation and a lethargic state. This increase is associated with asevere edema as indicated by an important increase of the W/D ratio.Lung weight gain was greater in the LPS-vehicle group compared to theLPS-vehicle group, at 4 hours post-gavage of 72 hour time point.ABC294640, administered at 250 mg/kg demonstrated a reduction ofthrombosis—evidenced by a reduction in blood clot length, weight andtotal thrombus score.

Example 3: Pilot Study—Treatment of COVID-19 with Pneumonia withOpaganib

Patients diagnosed with COVID-19 infection who have developed pneumoniaand do not require mechanical ventilation or who have been mechanicallyventilated for no more than 24 hours were assessed om this hospitalizedstudy.

Primary Objectives:

-   1) To evaluate the safety and tolerability of opaganib dosed at 500    mg Q12 hours in patients hospitalized with COVID-19 infection    To evaluate viral shedding on opaganib treatment

Secondary/Exploratory May Include One or More of:

-   1) To evaluate vital signs in patients hospitalized with COVID-19    infection on opaganib treatment-   2) To evaluate clinical improvement in patients hospitalized with    COVID-19 infection on opaganib treatment-   3) To evaluate the need for mechanical ventilation on opaganib    treatment, for patients not mechanically ventilated at baseline-   4) To evaluate the improvement in hypoxia either via SpO2/FiO2 or    PaO2/FiO2 ratio, and SpO2 on room air. Return to room air or a    specific SpO2 oxygen saturation on room air.    Assessment of on-treatment viral load changes, and changes in    D-dimer, cardiac troponin, LDH and ferritin levels.

Study Design:

This study included one active treatment arm; open-label opaganib 500 mgQ12 hour twice daily, for all eligible patients hospitalized withCOVID-19 pneumonia who either do not require mechanical ventilation, orhave received mechanical ventilation for <24 hours. Patients entered ascreening period of up to 1 week. Eligible patients entered thetreatment period for up to 2 weeks. All participants were followed for 2weeks after their last dose of study drug, at the end of the 2 weektreatment period, or once they had 2 consecutive daily negative viralswabs for the COVID-19 virus or at after premature drug discontinuationprior to Day 14. The maximum duration of study participation was 35 days(7 weeks). Study participants received opaganib, 2×250 mg capsules (500mg) Q12 hours, administered daily for up to a total of 14 days (2 weeks)or until 2 consecutive daily nasopharyngeal viral swabs were negativefor COVID-19, whichever came first. Opaganib was administered with food(after a light to moderate meal) and followed by 240 mL (8 fluid ounces)of water. If the patient was only able to take opaganib through anaso-gastric tube, the contents of the capsule were suspended in 20 ccnormal saline solution and pushed through the naso-gastric tube andflushed adequately with sterile water. If the patient was beingtube-fed, opaganib was administered shortly after (approximately 15-30minutes) a tube feeding.

Key Inclusion Criteria:

-   -   1. Adult male or female ≥18 to ≤75 years of age, inclusive    -   2. Proven COVID-19 infection and pneumonia not requiring        mechanical ventilation or mechanically ventilated for no more        than 24 hours at time of informed consent    -   3. The patient, guardian or legally acceptable representative        has signed a written IRB-approved informed consent.

Key Exclusion Criteria:

-   -   1. New York Heart Association Class III or IV, cardiac disease,        myocardial infarction within the past 6 months, unstable        arrhythmia, or evidence of ischemia on ECG    -   2. Any co-morbidity that may add risk to the treatment in the        judgement of the investigator.    -   3. Pregnant (positive serum test) or nursing women    -   4. Unwillingness or inability to comply with procedures required        in this protocol.    -   5. AST (SGOT) or ALT (SGPT)>2.5× upper limit of normal (ULN)    -   6. Bilirubin >1.5×ULN (except where bilirubin increase is due to        Gilbert's Syndrome)    -   7. Serum creatinine >2.0×ULN    -   8. Absolute neutrophil count <1000 cells/mm³    -   9. Platelet count <75,000/mm³    -   10. Hemoglobin <8.0 g/dL    -   11. Currently taking warfarin, apixaban, argatroban or        rivaroxaban    -   12. Current drug or alcohol abuse

Study Assessments:

The following will be monitored daily (see Table 3):

-   -   Review of concomitant medications    -   Adverse Events    -   Physical exam    -   Vital signs (temperature, blood pressure, pulse rate,        respiratory rate and oxygen saturation by pulse oximeter)    -   Clinical symptoms (cough, dyspnea, nausea, vomiting, diarrhea)    -   Nasopharyngeal viral swab    -   Serum chemistry    -   CBC with differential    -   Chest X-ray    -   Urinalysis

TABLE 3 Schedule of Assessments; other possible assessments notmentioned below include collecting blood and stool samples anddetermining SARS-CoV-2 RNA levels using a quantitative RT-PCR assay.Daily assessments of viral load as long as subjects continue to shedviral RNA. Daily On- Treatment Screening Assessments Days RandomizationDays 1-14 Assessments −7 to −1 Day 0 inclusive¹ ICF signed XInclusion/exclusion criteria X Demographics; medical X and surgicalhistory Review concomitant X X X medication(s) Review of systems X XPhysical examination X X X Vital signs² X X X Clinical symptomevaluation³ Weight X Nasopharyngeal viral swab X X X and/ororopharyngeal swab 12-lead ECG X Chest X-ray X X X Serum chemistry X X XHematology (CBC) X X X Urinalysis X X X Serum pregnancy test⁴ X ¹dailyassessments to Day 14 or earlier, if 2 daily consecutive negative viralswabs for SARS- CoV-2 ²assess temperature, blood pressure, pulse rate,respiratory rate and oxygen saturation by pulse oximeter ³assess cough,dyspnea, nausea, vomiting, diarrhea ⁴women of childbearing potential;serum pregnancy test must be negative within 3 days prior torandomization

Dosage Forms and Modes of Administration:

Opaganib was supplied as Capsules 250 mg, containing 250 mg opaganibalong with excipients in white opaque hard gelatin capsules.Opaganib was administered orally (or via naso-gastric tube whereappropriate) as two capsules (500 mg) every 12 hours for up to 2 weeks.Each dose is administered with food or 15-30 minutes after tube-feeding,where appropriate.

Study Endpoints: Primary Safety Endpoints:

-   1) Adverse events, laboratory tests, physical examination and vital    signs-   2) The percentage of patients with 2 negative consecutive daily    nasopharyngeal viral swabs by Day 14 on opaganib treatment    Secondary/Exploratory endpoints:-   1) The percentage of patients demonstrating vital sign improvement    (based on improvements in one or more of the following: temperature,    heart rate, respiratory rate, oxygen saturation)-   2) The percentage of patients demonstrating clinical improvement    (based on improvements in one or more of the following symptoms:    cough, dyspnea, nausea, vomiting, diarrhea)    The percentage of patients, who were not mechanically ventilated at    baseline who do not require mechanical ventilation by the end of the    2-week off-study-drug follow-up

Results:

-   -   Results have been obtained from seven patients approved for        compassionate use. These patients had moderate to severe        COVID-19-related pneumonia with hypoxia on supplemental oxygen.        The patients were given opaganib plus standard-of-care,        including hydroxychloroquine (HCQ) as background therapy in six        of the seven patients.    -   As can be seen from Table 4, with the exception of patient #7        who had only 1 day of treatment due to diarrhea that may or may        not be related to opaganib (the patient was also given HCQ and        Azithromycin), all 6 moderate to-severe patients improved        significantly, with 5 patients back to breathing room air and 3        patients discharged from hospital.    -   All six patients have shown reduction in C-Reactive Protein        (CRP), all six patients also demonstrated measurable clinical        improvement, including reduced supplemental oxygenation and        higher lymphocyte counts.    -   All patients started for first 3 days at 250 mg opaganib Q12        hours and then increased to 500 mg opaganib Q12 hours.    -   Despite being in only six patients, these preliminary findings        show clinical improvement in the first COVID-19 patients treated        with opaganib, and provide preliminary support for the        tolerability of opaganib use in COVID-19 patients.

TABLE 4 Results from Study Patient (days on opaganib Most recenttreatment) Clinical Assessment Laboratory Baseline (date) Patient 1 (14d) Started as severe on maximal OptiFlow Lymphocytes 1.6 2.8 (17 Apr.2020) 3 Apr. 2020; RA by 16/4. As of 22/4, one negative 10³/mm³ viralswab CRP mg/L 15.1 1 (17 Apr. 2020) Patient 2 (14 d) Started as severe+,planning for intubation Lymphocytes 1.1 2.1 (17 Apr. 2020) 7 Apr. 2020;RA by 20 Apr. 2020, D/C on 10³/mm³ 22 Apr. 2020 CRP mg/L 14.2 4.8 (17Apr. 2020) Patient 3 (7 d) Moderate-severe, on OptiFlow 13 Apr. 2020; RALymphocytes 0.9 1.2 (17 Apr. 2020) by 17 Apr. 2020, D/C on 19 Apr. 202010³ /mm³ CRP mg/L 10.7 1.7 (17 Apr. 2020) Patient 4 (11 d) Startedsevere+ 70% OptiFlow on 13 Apr. 2020; Lymphocytes 1.2 1 (22 Apr. 2020)RA on 22 Apr. 2020 10³ /mm³ CRP mg/L 24.8 17.4 (22 Apr. 2020) Patient 5(2 d) Started moderate on 13 Apr. 2020, nasal cannula. Lymphocytes 1.1 *Dramatic improvement after one day on 10³ /mm³ opaganib; D/C on 14 Apr.2020 on RA CRP mg/L 11 * Patient 6 (5 d) Started severe, high OptiFlowon 19 Apr. 2020. Lymphocytes 1.35 1.96 (24 Apr. 2020) No HCQ due tostroke, pacemaker and prolonged 10³ /mm³ QTc at baseline; down to nasalcannulas on CRP mg/L 12.2 6.8 (24 Apr. 2020) 24 Apr. 2020 Patient 7 (1 dtreatment discontinued) Started severe on high OptiFlow on 19 Apr. 2020;on HCQ and azithromycin <24 hours at the time treatment with opaganibstarted. Diarrhea the next day, all 3 meds were stopped, followed byworsening SOB, on 75% OptiFlow. On steroids only as of 24 Apr. 2020 *Improved one day after initiation of therapy, discharged on room airwithout repeat blood work. RA—room air, D/C—discharged, SOB—shortness ofbreath

Five patients were included in the analysis, and for comparisonpurposes, we used a control group with same-sex, same-severity patients(baseline characteristics). Univariate comparisons between the groupswere performed with chi-square test for categorical variables and t-testor MannWhitney U-test for continuous variables, as appropriate. Timevariables were compared with Cox proportional hazard regression,adjusted for age and background illnesses. CRP and lymphocyte changeswere compared utilizing a repeated measures general linear model with aBonferroni correction for multiple comparison.

Patients treated with opaganib had significantly faster increase inlymphocyte count. All other clinical outcomes had a non-statisticallysignificant trend in favor of the treatment group: median time toweaning from high-flow nasal cannula (HFNC) was 10 and 15 days in casesvs. controls (HR=0.3, 95% CI: 0.07-1.7, p=0.2), time to ambient air was13 vs 14.5 days (HR=0.4, 95% CI: 0.15-1.5), none of the cases requiredmechanical ventilation compared with 33% of controls. In this smallcohort of severe COVID-19 patients, opaganib was safe and well toleratedwith improvement in both clinical and laboratory parameters in alltreated patients. The efficacy of opaganib for COVID-19 infection shouldbe further tested in randomized placebo-controlled trials.

Example 4: Randomized, Double-Blind, Placebo-Controlled Phase 2a Studyof Opaganib in COVID-19 Pneumonia Primary Objective:

To evaluate the total oxygen requirement (area under the curve) usingdaily supplemental oxygen flow (L/min) over 14 days (Day 1 to Day 14)

Secondary Objectives:

-   1) To evaluate the time to 50% reduction from baseline in    supplemental oxygen based on oxygen flow in L/min-   2) To evaluate the proportion of patients no longer requiring    supplemental oxygen for at least 24 hours by Day 14-   3) To evaluate the proportion of afebrile patients at Day 14-   4) To evaluate the time to negative swabs for SARS-CoV-2 by PCR-   5) To evaluate the proportion of patients with negative swabs for    SARS-CoV-2 by PCR at Day 14-   6) To evaluate the need for intubation and mechanical ventilation by    Day 14-   7) To evaluate the time to mechanical ventilation-   8) To evaluate the proportion of patients, with at least one    measurement of fever at baseline (defined as temperature >38.0 C    [100.4 F]), who are afebrile (defined as temperature <37.2 C [99 F])    at Day 14-   9) To evaluate mortality 30 days post-baseline

Exploratory Objectives:

To assess the change in systemic markers of inflammation (D-dimer,cardiac troponin, C-reactive protein [CRP], lactate dehydrogenase [LDH]and ferritin)

Safety Objectives:

To assess the safety and tolerability of opaganib administered orally at500 mg Q 12 hours, for up to 14 days, in patients with COVID-19pneumonia

Study Population:

The study population will consist of patients diagnosed with COVID-19infection who have developed pneumonia defined as radiographic opacitieson chest X-ray and require supplemental oxygen. The patients must behospitalized at least during screening and at baseline (Day 1).

Study Design and Description:

This was a phase 2a, proof of concept, multi-center randomizeddouble-blind, parallel arm, placebo-controlled study. After informedconsent was obtained, patients entered a screening phase for no morethan 3 days, to determine eligibility. 42 eligible patients wererandomized to receive either opaganib added to standard of care, ormatching placebo added to standard of care, in a randomization ratio of1:1. Treatment assignments remained blinded to the patient, investigatorand hospital staff, as well as the sponsor. As there was no consensusfor a definitive treatment specifically targeting the SARS-CoV-2 viruscausing COVID-19 (Wilson, 2020), standard of care referred to regional,institutional or physician directed therapies, that were implementedduring the COVID-19 pandemic.

Study participants received either opaganib 2×250 mg capsules (500 mg)every 12 hours, or matching placebo, in addition to standard of care(pharmacological and/or supportive). Study drug was to be administeredevery day for 14 days (Day 1 to Day 14), unless the patient had beendischarged from the hospital without requiring supplemental oxygen, inwhich case study drug would only be administered to Day 10.

All participants were followed up for 4 weeks after their last dose ofstudy drug, which may have occurred at the end of the 2-weekdouble-blind treatment phase or after premature study drugdiscontinuation, based upon patient or physician determination. Themaximum duration of study participation was up to 45 days (including upto 3 days screening; 2 weeks DB treatment phase and 4-weeks off-studydrug follow-up)

Stratification:

Patients were stratified based on a minimization algorithm taking thefollowing three parameters into account: age at screening, ≥70 years ofage, (yes or no); HbA1c at screening, ≥6.5, (yes or no); oxygenrequirement at baseline, requiring non-invasive positive pressureventilation (e.g. via BIPAP, CPAP), (yes or no).

Eligibility Criteria: Inclusion:

-   1. Adult male or female ≥18 to ≤80 years of age-   2. Proven COVID-19 infection per RT-PCR assay of a pharyngeal sample    (nasopharyngeal or oropharyngeal) AND pneumonia defined as    radiographic opacities on chest X-ray-   3. The patient requires supplemental oxygen at baseline-   4. The patient, guardian or legal representative has signed a    written IRB-approved informed consent

Exclusion:

-   1. Any co-morbidity that may add risk to the treatment in the    judgement of the investigator.-   2. Requiring intubation and mechanical ventilation-   3. Oxygen saturation ≥95% on room air-   4. Any preexisting respiratory condition that requires intermittent    or continuous ambulatory oxygen prior to hospitalization-   5. Patient is, in the investigator's clinical judgement, unlikely to    survive >72 hours-   6. Pregnant (positive serum test within 3 days prior to    randomization) or nursing women-   7. Unwillingness or inability to comply with procedures required in    this protocol.-   8. Corrected QT (QTc) interval on electrocardiogram (ECG)>470 ms for    females or >450 ms for males, calculated using Friedericia's formula    (QTcF)-   9. AST (SGOT) or ALT (SGPT)>2.5× upper limit of normal (ULN)-   10. Bilirubin >1.5×ULN (except where bilirubin increase is due to    Gilbert's Syndrome)-   11. Serum creatinine >2.0×ULN-   12. Absolute neutrophil count <1000 cells/mm³-   13. Platelet count <75,000/mm³-   14. Hemoglobin <8.0 g/dL-   15. Currently taking medications that are sensitive CYP3A4, CYP2C9    or CYP2C19 substrates and have a narrow therapeutic index-   16. Currently taking medications that are strong inducers or    inhibitors of CYP2D6 and CYP3A4-   17. Currently taking warfarin, apixaban, argatroban or rivaroxaban-   18. Current drug or alcohol abuse-   19. Currently participating in a clinical study assessing    pharmacological treatments, including anti-viral studies

Number of Subjects:

A total of 49 patients were screened in the study, of which 42 patientswere randomized (23 to opaganib, 19 to placebo) while 7 were screenfailures. Two patients were randomized in each group but not treated. 19opaganib patients and 16 placebo patients completed treatment (Day 14).3 opaganib patients and 2 placebo patients discontinued the treatmentprematurely. Two patients in the opaganib arm experienced adverse eventssuch that study drug was terminated, while one placebo patient wasterminated due to an adverse event.

Screening/Baseline Assessments:

-   -   Signed informed consent    -   Eligibility determination    -   Complete medical history (including onset of COVID-19 symptoms)    -   Concomitant medication assessment    -   Baseline review of systems    -   Physical examination    -   Vital signs (temperature, blood pressure, pulse rate,        respiratory rate and oxygen saturation by pulse oximeter)    -   Weight if the patient is ambulatory    -   Oxygen requirement (L/min)    -   12-lead electrocardiogram    -   Chest Xray    -   Nasopharyngeal or oropharyngeal swab for SARS-CoV-2 PCR test    -   Serum chemistry    -   CRP, D-Dimer, LDH, ferritin, cardiac troponin    -   HbA1c    -   CBC with differential    -   Urinalysis    -   Serum pregnancy test (for women of childbearing potential)        within 3 days prior to treatment

Study Assessments:

The following were monitored and documented daily as part of thestandard of care:

-   -   Concomitant medications    -   Adverse Events    -   Interim Physical exam    -   Vital signs (temperature, blood pressure, pulse rate,        respiratory rate and oxygen saturation by pulse oximeter)    -   Oxygen requirement (L/min)        The following will be monitored less frequently as part of        standard of care and wherever possible:    -   For patients on concomitant hydroxychloroquine, a 12-lead        electrocardiogram (if allowed by hospital treatment guidelines        under COVID-19) approximately 3 hours after the first study drug        administration on Day 1, anytime on Days 2 and 4, and again at        end-of-treatment (either Day 10, 14 or at premature study drug        discontinuation). If patients are on monitors (including        telemetry or Holter monitors), investigators are encouraged to        collect QT interval data    -   Nasopharyngeal or oropharyngeal viral swab for SARS-CoV-2 PCR        test every 1-3 days    -   Serum chemistry once weekly    -   Serum CRP, D-Dimer, LDH, ferritin, cardiac troponin once weekly    -   CBC with differential once weekly    -   Chest X-ray as per physician decision

Study Endpoints: Primary

The total oxygen requirement (area under the curve) using the dailysupplemental oxygen flow (L/min) over 14 days (Day 1 to Day 14)

Secondary

-   1) Time to 50% reduction from baseline in supplemental oxygen based    on oxygen flow in L/min-   2) The percentage of patients no longer receiving supplemental    oxygen for at least 24 hours by Day 14-   3) The time to two consecutive negative swabs for SARS-CoV-2 by PCR,    at least 24 hours apart-   4) The percentage of patients with at least two consecutive negative    swabs, followed by continued negative swabs, for SARS-CoV-2 by PCR    at Day 14-   5) The percentage of patients requiring intubation and mechanical    ventilation by the end of the 2-week off-study-drug follow-up-   6) The time to intubation and mechanical ventilation-   7) The percentage of patients with at least one measurement of fever    at baseline (defined as temperature >38.0 C [100.4 F]), who are    afebrile (defined as temperature <37.2 C [99 F]) at Day 14-   8) Mortality due to any cause at Day 30

Exploratory

-   1) The mean change in systemic markers of inflammation (D-dimer,    cardiac troponin, C-reactive protein [CRP], procalcitonin [PCT],    lactate dehydrogenase [LDH] and ferritin) from baseline at Day

Safety

-   1) Incidence rates of all treatment-emergent AEs (TEAEs) and SAEs-   2) Evaluation of vital signs-   3) Evaluation of laboratory parameters (chemistry and hematology)-   4) Evaluation of electrocardiograms (ECG)

Statistical Methods:

The primary efficacy objective of the study was to evaluate the effectof Opaganib on total supplemental oxygen requirement (area under thecurve) using daily oxygen flow (L/min) measurements for 14 days (Day 1to Day 14). The primary efficacy endpoint calculated for each patientthe area under the curve of the supplemental oxygen requirement throughday 14, using the trapezoidal rule after subtracting the baseline oxygenrequirement at each day. Days where no supplementary oxygen was needed,were recorded as 0. If several values of oxygen requirement (L/min) arerecorded in a certain day, for the primary analysis the highest of thesevalues were taken. In the primary analysis, for patients who die beforeDay 14, or require intubation and mechanical ventilation, missing dailyvalues were assigned the maximal supplemental oxygen flow requirement of8 L/min. For patients discharged from hospital on supplemental oxygenprior to Day 14, if no values were collected by the site afterdischarge, the oxygen requirement (L/min) on the day of discharge wereto be assigned thereafter for each day to Day 14.

The primary analysis was based on the modified Intent to treatpopulation (mITT), which consist of all patients that were randomizedand treated with at least one dose of study drug (the populationincluded a total of 40 subjects, 22 in opaganib and 18 in placebogroup), Descriptive statistics of the baseline-adjusted AUC arepresented by group along with 95% confidence interval for each group andfor the difference in means between the groups. Supplemental oxygenrequirement up to Day 14 was collected even if a patient discontinuedtreatment prior to Day 14 but continued in the study to Day 14. Further,it was assumed that loss to follow up such that vital status up to Day14 were missing is unlikely. Therefore, the primary analysis assumedthat in case that all supplemental oxygen values are missing aftertreatment discontinuation, the last value is carried forward—until Day14, or death, if occurred before. A sensitivity analysis to the abovemissing data handling approach was performed using an AUC summarystatistics approach, in which groups AUC is calculated from theestimated parameters of a Repeated-Measures model.

Within the mITT cohort, two subjects withdrew their consent due to grade1 gastrointestinal AEs. In addition, one subject did not require anysupplemental oxygen at baseline prior to initiating treatment and wasremoved from several analyses as mandated in the statistical analysisplan (SAP). Thus, the post-hoc activity analysis population (“mITTsensitivity”) excluded these three patients from the analysis andincluded 37 subjects, 19 in opaganib and 18 in placebo. The results forboth the mITT population (which included these 3 subjects) and the mITTsensitivity population showed similar trends of activity.

Results:

Top-line results from the study found opaganib to be safe, with nomaterial safety differences between the opaganib and placebo treatmentarms. Overall, fewer patients suffered from serious adverse events(SAEs) in the opaganib treatment arm than in the placebo arm. In thissmall sample size, there were few events of intubation or fatality andthese were balanced between the two arms.

The opaganib-treated arm demonstrated a consistent trend of greaterimprovement in reducing oxygen requirement by end of treatment on Day 14across key primary and secondary efficacy outcomes, correlating withclinical improvement as defined by the World Health Organization (WHO)ordinal scale:

A greater improvement in the proportion of patients reaching room airand no longer requiring oxygen support by Day 14 vs. the control arm(52.6% vs. 22.2%). FIG. 4 shows a Kaplan-Meier curve of time to nolonger receiving supplemental oxygen for at least 24 hours (mITTsensitivity).

A greater improvement in the proportion of patients with 50% reductionin supplemental oxygen by day 14 vs. the control arm (89.5% vs. 66.7%).FIG. 5 shows a Kaplan-Meier curve of time cumulative incidence for timeto 50% reduction from baseline in supplemental oxygen based on oxygenflow in L/min (mITT sensitivity).

A higher proportion of patients discharged by Day 14 vs. the control arm(73.7% vs. 55.6%).

Greater reduction from baseline of the medial total oxygen requirement(AUC) over 14 days (68.0% vs. 46.7%). FIG. 6 shows a dot plot of totalsupplemental oxygen requirement (area under the curve) for percentchange from baseline using daily oxygen flow (L/min) measurements for 14days (day 1 to day 14).

Example 5: Randomized, Double-Blind, Placebo-Controlled InternationalPhase 2/3 Study—Opaganib in COVID-19 Pneumonia Primary Objective:

To evaluate the proportion of patients requiring intubation andmechanical ventilation by Day 14. The proportion of patients reachingroom air by Day 14.

Secondary Objectives:

-   1) To evaluate change on the WHO Ordinal Scale for Clinical    Improvement-   2) To evaluate the time to intubation and mechanical ventilation-   3) To evaluate the time to low oxygen flow via nasal cannula e.g.    from high oxygen flow via nasal cannula or CPAP, if high oxygen flow    is not an available option-   4) To evaluate the proportion of patients no longer requiring    supplemental oxygen for at least 24 hours by Day 14-   5) To evaluate the total oxygen requirement (area under the curve)    using daily supplemental oxygen flow (L/min) over 14 days (Day 1 to    Day 14)-   6) To evaluate the time to two consecutive negative swabs for    SARS-CoV-2 by PCR-   7) To evaluate the proportion of patients with two consecutive    negative swabs for SARS-CoV-2 by PCR at Day 14-   8) To evaluate the proportion of patients, with at least one    measurement of fever at baseline (defined as temperature >38.0 C    [100.4 F]), who are afebrile (defined as temperature <37.2 C [99 F])    at Day 14-   9) To evaluate mortality 30 days post-baseline

Explorative Objectives:

To assess the change in systemic markers of inflammation (D-dimer,cardiac troponin, C-reactive protein [CRP], lactate dehydrogenase [LDH]and ferritin) over the treatment period of 14 days.

Safety Objectives:

To assess the safety and tolerability of opaganib administered orally at500 mg Q 12 hours, for up to 14 days, in patients with severe COVID-19pneumonia.

Study Population:

The study population consisted of patients diagnosed with COVID-19infection that was defined as severe based on eligibility criteria toalign with current region-specific diagnostic guidance. Specifically,patients had at minimum pneumonia secondary to SARS-CoV-2, radiographicevidence of pneumonia on chest X-ray or CT scan, and requiredsupplemental oxygen by high flow oxygen via nasal cannula, face mask orCPAP, if high oxygen flow was not an available option. Patients werehospitalized at least during screening and at Baseline (Day 1).

Study Design and Description:

This was a phase 2/3 multi-center randomized, double-blind, parallelarm, placebo-controlled study with an adaptive design that utilized afutility assessment. The study was conducted in 40 sites in 8 countries,including Europe, Israel, Latin America (was approved also in US andUK).

After informed consent was obtained, patients entered a screening phase(no more than 3 days), to determine eligibility. 475 patients wererandomized and received either opaganib added to standard of care, ormatching placebo added to standard of care, in a randomization ratio of1:1. Treatment assignments remained blinded to the patient, investigatorand hospital staff, as well as the sponsor. As the approval and/orguidance for treating COVID-19 was evolving, for this protocol, standardof care was defined by the recommended schemes of treatment according tothe severity of the disease based on local diagnostic and guidelinedocuments such as the Temporary Methodic Recommendations: Prophylactic,Diagnostics and Treatment of New Corona Virus Infection (COVID-19); theEU Commission, the European Medicines Agency (EMA), the Heads ofMedicines Agency (HMA) and FDA, and as updated to the most currentversion of the recommendations.

Study participants received either opaganib 2×250 mg capsules (500 mg)every 12 hours, or matching placebo, in addition to standard of care(pharmacological as defined above and/or supportive) at any giveninstitution. Study drug was administered every day for 14 days (Day 1 toDay 14). All participants were followed up for 28 days after their lastdose of study drug, which occurred at Day 14 or after premature studydrug discontinuation, based upon patient or physician determination.

Randomization Strategy:

As the treatments in the recommended schemes of treatment according tothe severity of the disease differed, based on local diagnostic andguideline documents such as the Temporary Methodic Recommendations:Prophylactic, Diagnostics and Treatment of New Corona Virus Infection(COVID-19); the EU Commission, the European Medicines Agency (EMA), theHeads of Medicines Agency (HMA) and the FDA, standard of careadministered to patients differed by institution. In order to ensurebalance of standard treatment regimens in both treatment arms,randomization was determined at the individual site level.

Four independent data safety monitoring board (DSMB) recommendations tocontinue the study were received following three unblinded safetyreviews and an unblinded futility analysis be convened for the safetyoversight of the study in order to assuring safety of the trialparticipants.

Stratification:

Patients were stratified based on meeting three or more high riskclinical parameters for COVID-19 outcomes at baseline (yes or no). Theparameters were: 1) age at screening, ≥60 years of age, (yes or no); 2)male, (yes or no); 3) HbA1c at screening, ≥6.5 (yes or no); 4) hypoxemiawithout commensurate increased work of breathing (defined as increasedrespiratory rate, nasal flaring and/or increase use of respiratorymuscles including the diaphragm [yes or no]; 5) known underlying chroniclung disease (yes or no); 6) known cardiovascular disease orhypertension (yes or no); 7) BMI≥28.0 kg/m² (yes or no); 8) known renaldisease (yes or no).

Treatment and Administration:

Opaganib 500 mg Q12 hour or matching placebo. Opaganib or placebo madeinto a suspension form could be administered by nasogastric tube.

Study Duration:

The maximum duration of study participation was up to 45 days (includingup to 3 days screening; up to 14 days of double-blind treatment and 28days off-study drug follow-up).

Eligibility Criteria: Inclusion:

-   1. Adult male or female ≥18 to ≤80 years of age-   2. Proven COVID-19 infection per RT-PCR assay of a pharyngeal sample    (nasopharyngeal or oropharyngeal) AND pneumonia defined as    radiographic opacities on chest X-ray or CT scan-   3. The patient requires, at baseline, high flow supplemental oxygen    or CPAP, if high oxygen flow is not an available option (WHO Ordinal    Scale 5 at baseline).-   4. Patient agrees to use appropriate methods of contraception during    the study and 3 months after the last dose of study drug-   5. The patient or legal representative has signed a written informed    consent approved by the IRB/Ethics Committee

Exclusion:

-   1. Any co-morbidity that may add risk to the treatment in the    judgement of the investigator.-   2. Requiring intubation and mechanical ventilation-   3. Oxygen saturation ≥95% on room air-   4. Any preexisting respiratory condition that requires intermittent    or continuous ambulatory oxygen prior to hospitalization-   5. Patient is, in the investigator's clinical judgement, unlikely to    survive >72 hours-   6. Pregnant (positive serum or urine test within 3 days prior to    randomization) or nursing women.-   7. Unwillingness or inability to comply with procedures required in    this protocol.-   8. Corrected QT (QTc) interval on electrocardiogram (ECG)>470 ms for    females or >450 ms for males, calculated using Friedericia's formula    (QTcF)-   9. AST (SGOT) or ALT (SGPT)>2.5× upper limit of normal (ULN)-   10. Total bilirubin >1.5×ULN (except where bilirubin increase is due    to Gilbert's Syndrome)-   11. Serum creatinine >2.0×ULN-   12. Absolute neutrophil count <1000 cells/mm³-   13. Platelet count <75,000/mm³-   14. Hemoglobin <8.0 g/dL-   15. Currently taking medications that are sensitive CYP3A4, CYP2C9    or CYP2C19 substrates and have a narrow therapeutic index-   16. Currently taking medications that are strong inducers or    inhibitors of CYP2D6 and CYP3A4-   17. Currently taking warfarin, apixaban, argatroban or rivaroxaban    due to drug-drug interaction based on CYP450 metabolism-   18. Current drug or alcohol abuse-   19. Currently participating in a clinical study assessing    pharmacological treatments, including anti-viral studies

Screening/Baseline Assessments:

-   -   Signed informed consent by patient or legal representative    -   Eligibility determination    -   Complete medical history (including onset of COVID-19 symptoms)    -   Concomitant medication assessment    -   Baseline review of systems    -   Physical examination    -   Vital signs (temperature, blood pressure, pulse rate,        respiratory rate and oxygen saturation by pulse oximeter)    -   Weight if the patient is ambulatory    -   Oxygen requirement (L/min)    -   FiO2 (estimate)    -   12-lead electrocardiogram    -   Chest Xray or CT scan    -   Nasopharyngeal or oropharyngeal swab for SARS-CoV-2 PCR test    -   Serum chemistry    -   CRP, D-Dimer, LDH, ferritin, cardiac troponin    -   HbA1c    -   CBC with differential    -   Urinalysis    -   Serum or urine pregnancy test (for women of childbearing        potential) within 3 days prior to treatment

Study Assessments:

The following were monitored and documented daily as part of thestandard of care:

-   -   Concomitant medications    -   Adverse Events    -   Interim Physical exam    -   Vital signs (temperature, blood pressure, pulse rate,        respiratory rate and oxygen saturation by pulse oximeter)    -   Oxygen flow rate setting (L/min)    -   FiO2 (estimate or known if patient is ventilated)        The following was monitored less frequently as part of standard        of care and wherever possible:    -   For patients on concomitant        chloroquine/hydroxychloroquine/mefloquine, a 12-lead        electrocardiogram (if allowed by hospital treatment guidelines        under COVID-19) approximately 3 hours after the first study drug        administration on Day 1, anytime on Days 2 and 4, and again at        end-of-treatment (either Day 14 or at premature study drug        discontinuation). If patients were on monitors (including        telemetry or Holter monitors), investigators were encouraged to        collect QT interval data    -   Nasopharyngeal or oropharyngeal viral swab for SARS-CoV-2 PCR        test every 3 days    -   Serum chemistry once weekly    -   Serum CRP, D-Dimer, LDH, ferritin, cardiac troponin once weekly    -   CBC with differential once weekly    -   Chest X-ray or CT scan as per physician decision

Study Endpoints: Primary

The percentage of patients requiring intubation and mechanicalventilation by Day 14

Secondary

-   1) The percentage of patients with ≥2 category improvement on the    WHO Ordinal Scale for Clinical Improvement-   2) The time to intubation and mechanical ventilation-   3) The time to low oxygen flow via nasal cannula e.g. from high    oxygen flow via nasal cannula or CPAP, if high oxygen flow was not    an available option-   4) The percentage of patients no longer receiving supplemental    oxygen for at least 24 hours by Day 14-   5) The total oxygen requirement (area under the curve) using the    daily supplemental oxygen flow (L/min) over 14 days (Day 1 to Day    14)-   6) The time to two consecutive negative swabs for SARS-CoV-2 by PCR,    at least 24 hours apart-   7) The percentage of patients with at least two consecutive negative    swabs for SARS-CoV-2 by PCR at Day 14-   8) The percentage of patients with at least one measurement of fever    at baseline (defined as temperature >38.0 C [100.4 F]), who are    afebrile (defined as temperature <37.2 C [99 F]) at Day 14-   9) Mortality due to any cause at Day 30 after baseline

Exploratory

The mean change in systemic markers of inflammation (D-dimer, cardiactroponin, C-reactive protein [CRP], procalcitonin [PCT], lactatedehydrogenase [LDH] and ferritin) from baseline at Day 14

Safety

-   1) Incidence rates of all treatment-emergent AEs (TEAEs) and SAEs-   2) Evaluation of vital signs-   3) Evaluation of laboratory parameters (chemistry and hematology)-   4) Evaluation of electrocardiograms (ECG)

Prohibited Medications During the Study:

The following medications were prohibited during the study, includingthe 28-day follow-up period:

-   -   Medications that are sensitive CYP3A4, CYP2C9 or CYP2C19        substrates and have a narrow therapeutic index were prohibited    -   Strong inducers or inhibitors of CYP2D6 and 3A4 were prohibited        Warfarin, apixaban, argatroban and rivaroxaban were prohibited        due to drug-drug interaction based on CYP450 metabolism

Stopping Rules:

At any time during the study, participants stopped study drug if it wasdetermined that they have experienced any of the following adverseevents (using Grading criteria as defined in the revised NCI CommonTerminology for Adverse Events [CTCAE v.5.0])

-   -   Any neuropsychiatric adverse event of Grade 3 severity    -   Hallucinations of any severity (any Grade)    -   Nausea of Grade 3 severity    -   Vomiting of Grade 3 severity    -   Creatinine increase of Grade 2 severity

Statistical Methods:

The primary analysis was based on a composite failure (Yes/No) variable,indicating if a subject had required intubation and mechanicalventilation or had died by study Day 14.

In the rare case of unknown patient outcome (patient lost to follow up),it was also counted as treatment failure for the primary analysis. If apatient initiated new investigational therapy for COVID-19 within 14days, this was also regarded, in the primary analysis, as treatmentfailure.

The number and percentages of subjects with failure event was tabulatedper treatment group. A 95% confidence interval was constructed for eachproportion. A Cochran Mantel-Haenzel (CMH) test compared the proportionof failure between the two groups, using the study stratificationfactors used for randomization, and corresponding risk differenceestimate presented with 95% confidence interval. Exact confidenceintervals were used as needed.

The significance level for this test was two-sided 5%. In the case ofsmall number of events (less than 5 events in any study arm), the Fisherexact test was used.

The number and percent of each of the failure types (intubation andmechanical ventilation was described by group.

The primary analysis was based on the modified Intent to treatpopulation (mITT), which consisted of all patients that were randomizedand treated with at least one dose of study drug. Analysis demonstrateda compelling benefit of opaganib in the ‘moderately severe’ patients inthe study:

-   -   These were 251/463 subjects (54% of mITT) requiring Fraction of        inspired Oxygen (FiO2) up to and including 60% at baseline        (median FiO2)    -   These patients represent the lower half of the study's        population in terms of oxygen requirement & disease severity        (based on the median)    -   This covers all the low-flow patients (WHO Ordinal Scale 4) and        approximately half of the high-flow patients (WHO Ordinal Scale        5)—together these are the vast majority of hospitalized COVID-19        patients    -   Consistent with prior U.S. Phase 2 study results presented in        Example 4 (majority of low flow patients) and opaganib's        anti-viral effect, the FiO2 up to 60% parameter is highly        informative for directing opaganib to this patient population        that stands to significantly benefit from the treatment and        represents a paradigm changing approach to classifying disease        severity in COVID-19.    -   Mortality: Opaganib treatment resulted in a significant 62%        reduction in mortality (7/117 patients treated with opaganib vs.        21/134 for placebo; nominal p-value=0.019, Relative Risk 2.6)        (Sensitivity Analysis: 5/117 vs. 16/134, a significant 64%        efficacy benefit; nominal p-value=0.033, Relative Risk—2.8), see        FIG. 7    -   Reaching Room Air by Day 14 (primary endpoint of the study):        90/117 (77%) of opaganib-treated patients reached room air by        Day 14 vs. 85/134 (63.5%) for placebo—a significant efficacy        benefit of 21% with opaganib (nominal p-value=0.033 by Cochran        Mantel-Haenzel test using the study stratification factors used        for randomization, and corresponding stratified proportion        difference with 95% CI). (No longer received supplemental oxygen        for at least 24 hours by Day 14)    -   Median time to discharge: Patients treated with opaganib showed        median time of 10 days to discharge vs. 14 days for the placebo        arm, resulting in a significant saving of four days        hospitalization per opaganib patient (nominal p-value=0.0195),        see FIG. 8 .    -   Safety: Overall adverse events were balanced between the        opaganib and placebo groups, suggesting good safety, with no new        safety signals emerging

TABLE 5 Benefit observed across secondary endpoints with nominallysignificant p-values: Opaganib Placebo Parameter Statistics (N = 117) (N= 134) Outcome Patients With an Improvement of 2 n (%) 93 (79.49) 88(65.67) or More on the WHO Ordinal Scale Compared to Baseline by Day 14*Difference in Rates (Opaganib - % 13.82 Placebo) p-value** 0.023 Time toa score of <=3 on the WHO Ordinal Scale Number of Events n (%) 93 (79.5)88 (65.7) Kaplan-Meier Median (days) Estimate 95% CI 8.00 10.00(7.00-9.00) (9.00-12.00) p-value** 0.010 Time to Discharge by day 42Number of Events (%) n (%) 81 (69.2) 85 (63.4) Restricted Mean Analysisp-value 0.0022 Kaplan-Meier Mean (days) Estimate 13.41 17.89 Difference(days) and 95% CI −4.48 (−7.34, −1.62) *Analysis statistics wereestimated using Log Rank test stratified by study stratification factorsused for randomization. A negative (positive) statistic is associatedwith longer (shorter) time to the event **p-value from CochranMantel-Haenzel test using the study stratification factors used forrandomization, and corresponding stratified proportion difference with95% CI Evaluation of baseline biomarkers supports the use of FiO2 asindicator of disease severity and as a predictor of the treatmentbenefit in the target population.

TABLE 6 Biomarker medians by subpopulations (FiO2 ≤ 60% and FiO2 ≥ 60%at baseline) Lower FiO2 Higher FiO2 Marker N Median Q1, Q3 N Median Q1,Q3 p-value* Lymphocytes 246 0.990 0.70, 1.38 178 0.780 0.50, 1.16 <.0001(10⁹/L) C Reactive Protein 240 60.800 21.40, 153.40 186 102.800 38.67,200.30 0.0005 (mg/L) Ferritin (μg/L) 228 666.950 370.17, 1297.50 1671000.000 500.60, 2000.00 0.0008 D-Dimer (μg/ml) 240 0.450 0.18, 1.06 1780.806 0.31, 1.79 0.0004 Lactate 230 361.150 290.60, 522.00 183 469.230344.00, 644.00 <.0001 Dehydrogenase (IU/L) Troponin T (μg/L) 44 0.0090.00, 0.06 60 0.010 0.01, 0.02 0.1616 *Comparing opaganib vs. placeboarms for imbalances

The post-hoc analysis is robust, rigorous and adheres to strictstandards of integrity and reliability. FiO2 is a clinically andmedically relevant parameter for oxygen requirement and diseaseseverity, irrespective of the device providing the oxygen. The cutoffselected of FiO2 up to 60% was objectively based on the median of thedistribution of FiO2 levels globally across the entire study and acrossa wide range of oxygen supplementation devices. Potential cofoundingfactors were rigorously analyzed—negating potential impact of baselinerisk-factors and SOC on the outcome. Territorial variability wasanalyzed—showing robust consistency.

INDUSTRIAL APPLICABILITY

The present invention provides an anti-coronavirus agent comprising asan active ingredient a compound represented by:

as a free base or as a salt thereof, an anti-SARS agent comprising theanti-coronavirus agent and a method of treating SARS using theanti-coronavirus agent. The present invention enables the treatment ofdiseases caused by coronaviruses, especially the SARS-associatedcoronavirus.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. Variousmodifications and variations of the described compositions and methodsof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific embodiments, itwill be understood that the invention should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the fields of molecular biology, medicine, immunology,pharmacology, virology, or related fields are intended to be within thescope of the invention.

What is claimed is:
 1. A method of determining the efficacy of a treatment for coronavirus pneumonia in a patient, the method comprising: detecting a concentration of a biomarker in inhaled gas given to the patient prior to the treatment, wherein the biomarker is a molar or volumetric fraction of inspired oxygen (FiO2) and the concentration is up to and including 60% supplemental oxygen; administering the treatment to the patient to wean the concentration of supplemental oxygen down to a lower concentration while still providing adequate oxygenation to the patient, wherein the treatment comprises administering an effective amount of ABC294640,

as a free base or salt thereof to the patient; detecting the concentration of the biomarker in inhaled gas given to the patient after the treatment, wherein a decrease in the concentration of the biomarker after the treatment compared to the concentration of the biomarker prior to the treatment indicates efficacy of the treatment; and determining that the treatment is effective based on the concentration of the biomarker after treatment compared to the concentration of the biomarker before treatment.
 2. The method of claim 1, further comprising altering or continuing the treatment based on whether the treatment was effective at lowering the concentration of the biomarker.
 3. The method of claim 1, wherein ABC294640 is in the form of a free base.
 4. The method of claim 1, wherein ABC294640 is in the form of hydrochloride salt.
 5. The method of claim 4, further comprising a pharmaceutically-acceptable carrier material, wherein the ABC294640 hydrochloride and the pharmaceutically-acceptable carrier material are in a unit dosage form suitable for oral administration.
 6. The method of claim 5, wherein the unit dosage form is a solid dosage form.
 7. The method of claim 6, wherein the solid dosage form is a capsule.
 8. The method of claim 1, wherein the patient is receiving supplemental oxygen via noninvasive positive-pressure ventilation.
 9. The method of claim 8, wherein the patient is receiving supplemental oxygen via nasal cannula.
 10. The method of claim 8, wherein the patient is receiving supplemental oxygen via face mask.
 11. The method of claim 10, wherein the respiratory syndrome coronavirus is a severe acute respiratory syndrome coronavirus (SARS).
 12. The method of claim 11, wherein the severe acute respiratory syndrome coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
 13. The method of claim 12, wherein the SARS-CoV-2 virus is a naturally occurring coronavirus.
 14. The method of claim 12, wherein the SARS-CoV-2 virus is a naturally occurring coronavirus variant.
 15. The method of claim 4, wherein the unit dosage form suitable for oral administration is a capsule having 250 mg of ABC294640 hydrochloride, and wherein administering includes two capsules administered twice a day, for at least 10 days, for a total daily dose of 1000 mg of ABC294640 hydrochloride.
 16. The method of claim 3, further comprising a pharmaceutically-acceptable carrier material, wherein the pharmaceutically-acceptable carrier material is physiologically buffered saline.
 17. The method of claim 16, wherein a suspension is formed that includes ABC294640 hydrochloride suspended in physiologically buffered saline, and wherein the administering includes using a tube to deliver the suspension directly to the stomach.
 18. The method of claim 2, further comprising co-administering to the patient at least one additional antiviral active agent.
 19. The method of claim 18, wherein the antiviral active agent is a nucleotide-analog anti-viral prodrug.
 20. A test method for predicting therapeutic efficacy of and/or predicting prognosis in a coronavirus pneumonia patient requiring high flow supplemental oxygen or continuous positive airway pressure (CPAP) for administration of ABC294640 as a free base or salt thereof, comprising: measuring the molar or volumetric fraction of inspired oxygen (FiO2) in inhaled gas before initiation of therapy with ABC294640 and in an early phase after initiation of therapy with ABC294640; comparing the molar or volumetric FiO2 in the inhaled gas collected from the patient before initiation of therapy with the molar or volumetric FiO2 in the inhaled gas collected from the patient in the early phase after initiation of therapy; and determining the therapeutic efficacy and/or prognosis for administration is poor when a difference between the molar or volumetric FiO2 in inhaled gas collected from the patient in the early phase after initiation of therapy and the molar or volumetric FiO2 in inhaled gas collected from the patient before initiation of therapy is greater than a predetermined threshold difference 